Our results showed that ICV α7 nAch receptor agonists PHA decreased depression-like behaviors induced by SNI and CFA, the mechanisms could be due to the activation of WNT/β-catenin signal pathway and suppression of brain inflammation.
Pain and depression comorbidity have been observed in clinics and investigated in animal models. In the study, we found that SNI-induced depression-like behaviors after surgery. The results were confirmed by decreased center and center/total distance in the open field tests, and decreased sucrose and food intake, and weight gain compared to naïve rats. SNI surgery did not affect the total distance moved in the open field test, which excluded the possibility that the effect was attributed to motor impairment. Pain behaviors of paw withdrawal threshold and response to acetone suggest the successful establishment of pain animal models. In addition, animals received CFA injection, which is proinflammatory and induces pain, also present depressive behaviors, which confirm chronic pain induces depressive conditions. Clinical study has demonstrated that chronic pain frequently results in depression, and chronic pain contributed to the recurrence, longer duration and increased sadness of depressive episodes. In addition, pain and depression were intertwined and co-exacerbate physical and psychological symptoms. The mechanism may be explained by some overlap biological process. For example, injuries to sensory pathways have been demonstrated to affect the same brain regions that are involved in mood disorders.
Recently, depression has been shown to be an inflammatory disease, and inhibition of inflammation reduces depression-like behaviors. In clinical study, neuroinflammation of cytokines has been analyzed in CSF of post-mortem brain tissue, and results found immune cells are involved in the immune response using both postmortem tissue and positron emission tomography (PET) imaging.The specialized immune cells in the brain are called microglia that comprise 5%–10% of total brain cells and their main functions are to maintain CNS homeostasis and to cope with damage or infection. In many neurodegenerative and neuropsychiatric diseases, microglia are activated and contribute to pathology by promoting neuroinflammation. In depressive conditions, microglia are activated and induce cascade proinflammatory response in the brain and contribute to the neuropathology and depression-like behaviors. Evidence in rodents found that after insult, microglial indoleamine 2, 3-dioxygenase and kynurenine pathway are activated and generate excessive neurotoxic quinolinic acid, microglia shift to proinflammatory profile, and generates a host of cytokines, including interleukin IL-1β, TNF-α and so on. The proinflammatory quinolinic acid and cytokines from microglia inhibit hippocampal neurogenesis and induce depression-like behaviors, whereas treatment with minocycline, a microglia inhibitor, counteracted this effect. Thus, suppression of the brain microglia-related inflammation is effective approach to treating depression.
PHA is a α7 nAch receptor agonist, and our results showed that ICV injection of PHA decreased depression-like behaviors, including open field test, sucrose preference test and food intake induced by SNI, the reason could be attributed to the activation of α7 nAch receptors, as co-administration of α7 nAch receptor antagonist MLA abolished that effect. Consistently, our previous study showed that IT injection of PHA prevents the development and maintenance of chronic pain. The reason is attributed to suppression of proinflammatory response in the spinal cord. In addition, PHA decreased inflammatory cytokines release from microglia in vitro by activation of α7 nAch receptors in primary microglia cultures. Study have demonstrated that the inflammation in the brain majorly comes from microglia, as microglia suppression prevents the depression-like behaviors in animals. In our study, we included a group of rats received MIN, a microglia inhibitor, and results showed that ICV MIN decreased SNI-induced depression-like behaviors and brain proinflammatory response. This confirmed depression is a microglial inflammatory disease and suppression of microglia-related inflammation reversed depression-like behaviors induced by SNI, which is consistent with our previous study.
The cholinergic anti-inflammatory pathway controls cytokine production and inflammation in the body. Experimental evidence suggests that cholinergic anti-inflammatory signaling requires a7 nAch receptors expressed on non-neuronal cytokine-producing microglia in the central nervous system. a7 nAch receptor agonists inhibit cytokine release and shows protective effect in a variety of experimental lethal inflammatory models and has been exploited to counteract abnormal chronic and hyper-activated pro-inflammatory conditions. Our study confirmed that suppression of inflammation in the brain is a promising therapeutic target for treating depression by activation of a7 nAch receptors. In order to confirm that the effect of α7 nAch receptor activation is not limited to SNI-induced depression, we included CFA model to prove the general effect of α7 nAch receptor agonist in chronic pain-induced depression. Our results showed that CFA-induced depression-like behaviors, including decreased center distance, center/total distance, sucrose intake percentage and food intake, and ICV PHA provided anti-depressive effect induced by CFA. In addition, the anti-depressive effect of PHA was reversed by MLA, which confirmed that PHA’s anti-depressive effect by activation of α7 nAch receptors. Further, PHA decreased CFA-induced brain inflammation, including increased IL-10, decreased IL-1β, TNF-α and IL-18 in the brain.
To further explore the mechanisms of the anti-depressive effect of PHA, signal pathway inhibitors were administered at the same time. The results showed that WNT/β-catenin inhibitor IWR reversed the anti-depressive effect of PHA in the open field test and sucrose preference test, and both WNT/β-catenin inhibitor IWR and mTOR inhibitor suppressed food intake and weight gain, whereas TGF-β, Notch, and JAK pathway inhibitors did not affect that effect. This suggests the anti-depressive effect of PHA through WNT/β-catenin, while feeding behavior of food and weight gain are affected by both WNT/β-catenin and mTOR signal pathway. WNT/β-catenin are secreted glycoproteins that signals through the frizzled receptors and are essential for normal embryonic development[27, 28]. Study have demonstrated WNT/β-catenin signaling is associated in depression and antidepressant response. The WNT/β-catenin pathway in nucleus accumbens is down-regulated in the mouse model of social defeat model of depression, and knock down of the signaling renders mice more susceptible to social defeat stress and promotes depression-like behaviors. In addition, different classes of antidepressant treatments, including SSRIs, SNRI, and chronic electroconvulsive shock, showed anti-depressive effect by activation of WNT/β-catenin pathway. However, our study suggests activation of a7 nAch receptors showed anti-depressive effect via the WNT/β-catenin pathway.
Our result showed that the mTOR inhibitor decreased weight gain of PHA. the reason may be attributed to that mTOR controls the body metabolism and increases food intake and consequently result in weight gain. Thus, food intake and weight gain are also affected by mTOR inhibitors in our experiments. The inflammatory cytokine concentration was measured and the results showed WNT/β-catenin inhibitor IWR reversed the IL-10, IL-1β, TNF-α and IL-18 content change induced by PHA, which confirmed PHA present its anti-inflammation and anti-depressive effect via WNT/β-catenin signal pathway. Surprisingly, IL-1β, TNF-α content change were reversed also by mTOR pathway inhibitors. This indicates that mTOR pathway is also anti-inflammatory in chronic pain-induced depressive animal models. Although previous study has demonstrated that the activation of mTOR reversed depression-like behaviors, the mechanisms may be explained by suppression of inflammation[32, 33]. However, mTOR pathway inhibitor did not affect anti-depressive effect of PHA, and this suggested mTOR maybe a downstream signal of WNT/β-catenin, or at least not a major contributor of the anti-depressive effect of PHA. PHA activates WNT/β-catenin and results in the increment of IL-10 and the decrement of IL-1β, TNF-α, IL18 release, and at the same time, activates mTOR which result in enhanced decrement of IL-1β and TNF-α release. As previous study has demonstrated IL-10 can resolve the proinflammatory response, the anti-depressive effect of PHA should be attributed to the activation of WNT/β-catenin and increased IL-10 release.
In the study, several cytokines were chosen to evaluate the brain inflammatory response. Previous studies have demonstrated that IL-10, IL-1β, TNF-α and IL-18 were related to depression, and modulation of inflammation can induce or decrease depression-like behaviors. In our study, both SNI and CFA increased IL-1β, TNF-α and IL-18 in the brain, and ICV PHA increased IL-10, and decreased IL-1β, TNF-α and IL-18 content. This confirmed increased anti-inflammatory IL-10 suppressed pro-inflammatory response after chronic pain insult. In addition, followed our previous study, tissue of mPFC BA and VH were acquired to testify the inflammatory profile after SNI or CFA, because those sites have been proved to be involved in depression and showed increased cytokines expression after insult. Our research verified that these sites are proper and effective for the study of depression and anti-depressive treatment response.